Investigations on the assembly, structure, and physiology of the outer membrane of Escherichia coli will be continued as follows. It is possible to fractionate outer membrane fragments on the basis of their content of lipopolysaccharide by affinity chromatography on columns containing antibody specific for a given lipopolysaccharide. This method will be used to fractionate the previously detected specialized regions via which new lipopolysaccharide enters the outer membrane. The rate of diffusion of lipopolysaccharide from these points to the rest of the outer membrane will also be measured. By specificially labeling lipid, protein, and peptidoglycan, the relationship of the insertion points for lipopolysaccharide to the location of newly inserted other components will be measured. Previous work from this laboratory indicated that exposure to EDTA released half of the lipopolysaccharide with a small amount of phospholipid while leaving its structure intact. The structure of the cell membrane before and after EDTA treatment will be studied by electron spin resonance and by accessibility to immobilized enzymes such as proteases and lipases. Such studies will help define the location of the EDTA-released moiety with respect to other outer membrane components.